Sperm exocytosis reconstructed in a cell-free system: Evidence for the involvement of phospholipase c and actin filaments in membrane fusion

Abstract

We used a cell-free system to study membrane fusion during sperm exocytosis (acrosome reaction). Extracted bovine sperm plasma and outer acrosomal membranes were labeled with chlorophyll a or DCY, respectively. The occurrence of membrane fusion is indicated by the ability of the probes to diffuse from one membrane species to another which is revealed by resonance energy transfer between the two probes. We have previously shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that the pH and calcium dependence of the cell-free fusion mimics those of exocytosis in intact cells. In the present report we further characterize the fusion of sperm membranes which we observe in our assay. Phosphoproteins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and Gi-like proteins, while involved in exocytosis in intact cells, are not involved specifically in the membrane fusion step of exocytosis. The role of membrane bound F-actin in regulating membrane fusion was also studied using fluorescently labeled phalloidin. The results show that cortical F-actin has two roles in regulating sperm exocytosis. One is to form a scaffolding to hold phospholipase C at the membrane. It also functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion.

Origin of the Lag Period in the Phospholipase C Cleavage of Phospholipids in Membranes. Concomitant Vesicle Aggregation and Enzyme Activation

“…This was the first report of a model system in which fusion was induced by a catalytic agent. Other authors have pointed out the putative role of phospholipase C in physiological cell fusion events (Roldan & Harris, 1989;Liu et al, 1993;Spungin et al,1995). While the degree of implication of phospholipase C in cell membrane fusion remains to be established, the enzyme-driven liposome fusion process shows nevertheless remarkable regulatory properties.…”

“…Phospholipases C have been comparatively less studied, in spite of their essential role in several aspects of metabolic regulation (Berridge et al, 1987;Exton, 1990;Haines et al, 1992). Phospholipases C may equally be involved in membrane fusion events (Roldan & Harris, 1989;Liu et al, 1993;Spungin et al, 1995). Previous investigations from this laboratory have revealed that the catalytic activity of phospholipase C may promote phospholipid vesicle fusion (Nieva et al, 1989(Nieva et al, , 1993(Nieva et al, , 1995.…”

Dual Inhibitory Effect of Gangliosides on Phospholipase C-Promoted Fusion of Lipidic Vesicles

“…The inescapable question on the biological relevance of the above observations cannot receive a fully satisfactory answer as yet. However, phospholipase C (and Ca 2+ in millimolar concentrations) has been found to be involved in a physiological fusion process, namely, sperm exocytosis, and is probably important in other exocytotic processes (Rolda ´n & Harrison, 1989;Spungin et al, 1995). Moreover, the growing consensus on a single topological mechanism underlying the various fusion processes (see introduction) increases the validity and relevance of studies involving model systems.…”